Reactive oxygen species such as superoxide anion (O2.-), are well known mediators of endothelial dysfunction, especially during the early stages of atherosclerosis. Several lines of evidence strongly suggest that alterations in vascular fluid flow dynamics promotes the excess generation of endothelial O2.- which in turn, contributes to early atherosclerotic plaque formation. A clear link between endothelial O2.- production and the specific hemodynamic forces involved in endothelial O2.- production has not been well defined. To better understand the pathogenesis of early atherosclerosis, the goal of the proposed research is to elucidate the mechanism(s) by which specific flow profiles lead to the generation of O2.- in vascular endothelial cells. The over all specific aims of the project are: A) Identify the specific shear profiles that stimulate O2.- production in endothelial cells. B) Identify the oxidative enzymes responsible for shear induced endothelial O2.- production. Finally to better elucidate the molecular mechanism(s) involved in shear signal transduction, C) Assess the role of G proteins as a possible mediator of shear induced endothelial O2.- production. Unique to this study is the continuous chemiluminescent detection of endothelial O2.- production during exposure of the cells to the specific shear stress profiles. This project will provide strong evidence for the central role that fluid shear and O2.- play in the early pathogenesis of atherosclerosis. Furthermore, it will have broad application for the understanding of the molecular mechanism(s) by which flow is sensed and the signal transduced across the endothelial plasma membrane.